title: Evidence for two CRIB domains in phospholipase D2 (PLD2) that the enzyme uses to specifically bind to the small GTPase Rac2.
abstract: Phospholipase D (PLD) and small GTPases are vital to cell signaling. We report that the Rac2 and the PLD2 isoforms exist in the cell as a lipase-GTPase complex that enables the two proteins to elicit their respective functionalities. A strong association between the two molecules was demonstrated by co-immunoprecipitation and was confirmed in living cells by FRET with CFP-Rac2 and YFP-PLD2 fluorescent chimeras. We have identified the amino acids in PLD2 that define a specific binding site to Rac2. This site is composed of two CRIB (Cdc42-and Rac-interactive binding) motifs that we have named "CRIB-1" and "CRIB-2" in and around the PH domain in PLD2. Deletion mutants PLD2-ΔCRIB-1/2 negate co-immunoprecipitation with Rac2 and diminish the FRET signal in living cells. The PLD2-Rac2 association was further confirmed in vitro using affinity-purified recombinant proteins. Binding was saturable with an apparent K(d) of 3 nm and was diminished with PLD2-ΔCRIB mutants. Furthermore, PLD2 bound more efficiently to Rac2-GTP than to Rac2-GDP or to a GDP-constitutive Rac2-N17 mutant. Increasing concentrations of recombinant Rac2 in vitro and in vivo during cell adhesion inhibit PLD2. Conversely, Rac2 activity is increased in the presence of PLD2-WT but not in PLD2-ΔCRIB. We propose that in activated cells PLD2 affects Rac2 in an initial positive feedback, but as Rac2-GTP accumulates in the cell, this constitutes a "termination signal" leading to PLD2 inactivation.
authors.affiliation: Department of Biochemistry and Molecular Biology, Wright State University School Medicine, Dayton, Ohio 45435, USA.
authors.forename: Hong-Juan
authors.initials: HJ
authors.lastname: Peng
authors.forename: Karen M
authors.initials: KM
authors.lastname: Henkels
authors.forename: Madhu
authors.initials: M
authors.lastname: Mahankali
authors.forename: Mary C
authors.initials: MC
authors.lastname: Dinauer
authors.forename: Julian
authors.initials: J
authors.lastname: Gomez-Cambronero
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction / Q000502:physiology*; D018411:Spodoptera; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000097766:RAC2 GTP-Binding Protein
chemical_list: D007527:Isoenzymes; D010739:Phospholipase D; D011993:Recombinant Fusion Proteins; D020744:rac GTP-Binding Proteins; D000097766:RAC2 GTP-Binding Protein; C105606:phospholipase D2